Steroid hormones, thyroid hormones, retinoids, and vitamin D are small hydrophobic molecules that serve as important signaling molecules throughout the body. Although all of these molecules are insoluble in water, they are made soluble for transport in the bloodstream and other extracellular fluids by binding to specific carrier proteins, from which they dissociate before entering a target cell. One such carrier protein is transthyretin.
Transthyretin (also known as TTR; TTR, prealbumin; prealbumin, thyroxine; PALB; TBPA; HST2651; amyloidosis 1, included; dysprealbuminemic euthyroidal hyperthyroxinemia, included; hyperthytoxinemia, dysprealbuminemic, included; hyperthytoxinemia, dystransthyretinemic, included; amyloid polyneuropathy, multiple forms, included; senile systemic amyloidosis, included) is a homotetrameric transport protein found in the extracellular fluids of vertebrates (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).
Transthyretin was first identified as the major thyroid hormone carrier in the cerebrospinal fluid (CSF) and in the serum (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300; Seibert, J. Biol. Chem., 1942, 143, 29-38). Transthyretin was cloned from adult human cDNA libraries and the gene was subsequently mapped to chromosome region 18q11.2-q12.1 (Mita et al., Biochem Biophys Res Commun, 1984, 124, 558-564; Sparkes et al., Hum Genet, 1987, 75, 151-154; Whitehead et al., Mol Biol Med, 1984, 2, 411-423).
The liver and the choroid plexus are the primary sites of transthyretin synthesis in humans (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).
Transthyretin that is synthesized in the liver is secreted into the blood, whereas transthyretin originating in the choroid plexus is destined for the CSF. In the choroid plexus, transthyretin synthesis represents about 20% of total local protein synthesis and as much as 25% of the total CSF protein (Dickson et al., J Biol Chem, 1986, 261, 3475-3478). Transthyretin synthesis has also been identified in the yolk sac of developing rats (Soprano et al., Proc Natl Acad Sci USA, 1986, 83, 7330-7334); the retina, ciliar body and optic nerve regions of bovine and rat eyes (Martone et al., Biochem Biophys Res Commun, 1988, 151, 905-912; Ong et al., Biochemistry, 1994, 33, 1835-1842); human and porcine pancreatic islets (Jacobsson et al., J Histochem Cytochem, 1989, 37, 31-37) and, in minor amounts, in the stomach, heart, skeletal muscle, and spleen of rats (Soprano et al., J Biol Chem, 1985, 260, 11793-11798).
It is currently believed that transthyretin serves as a hormone reservoir. As demand for thyroid hormone increases, transthyretin increases the transport and release of hormone to targets such as brain, kidney, and cardiac tissues, thereby ensuring a uniform hormone distribution within the cells in each of these tissues (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300). Transthyretin transports the thyroid hormones triiodothyronine (T3) and thyroxine (T4) as well as the retinol/retinol-binding protein complex. A mouse strain deficient in transthyretin is viable and fertile, yet exhibits significantly depressed levels of serum retinol, retinol-binding protein, and thyroid hormone, confirming transthyretin's role in maintaining normal levels of these metabolites in circulating plasma (Episkopou et al., Proc Natl Acad Sci USA, 1993, 90, 2375-2379). In addition to serving as a transport protein, transthyretin has been reported to have a variety of other functions, including: inhibiting interleukin-1 production in monocytes and endothelial cells (Borish et al., Inflammation, 1992, 16, 471-484); involvement in the metabolism of the environmental pollutant polyhalogenated biphenyl (Brouwer and van den Berg, Toxicol Appl Pharmacol, 1986, 85, 301-312); and binding pterins (Emstrom et al., FEBS Lett, 1995, 360, 177-182). Furthermore, in recent years a link between transthyretin and lipoprotein biology has become increasingly apparent. A fraction of plasma transthyretin circulates in high density lipoproteins (HDL) through binding to apolipoprotein A-1 (Sousa et al., J Biol Chem, 2000, 275, 38176-38181), and transthyretin has been shown to proteolytically process apolipoprotein A-1 (Liz et al., J Biol Chem, 2004). Furthermore, transthyretin reabsorption by the kidneys is mediated by the lipoprotein receptor megalin (Sousa et al., J Biol Chem, 2000, 275, 38176-38181). This reabsorption serves as a means for preventing hormone loss in urine. Finally, the major site of degradation for both transthyretin and lipoproteins is the liver. There is considerable evidence that hepatic uptake of both transthyretin and lipoproteins is mediated by an as yet unidentified lipoprotein receptor, suggesting a shared degradation pathway (Sousa and Saraiva, J Biol Chem, 2001, 276, 14420-14425).
Transthyretin is associated with both local and systemic amyloidosis, a disorder characterized by extracellular systemic deposition of mutated or wild-type transthyretin as amyloid fibrils (Cornwell et al., Biochem Biophys Res Commun, 1988, 154, 648-653; Saraiva et al., J Clin Invest, 1984, 74, 104-119; Yazaki et al., Muscle Nerve, 2003, 28, 438-442), leading to organ dysfunction and death. Senile systemic amyloidosis is a sporadic disorder resulting from the extracellular deposition of wild-type transthyretin fibrils in cardiac and other tissues. Over 80 mutations in transthyretin are associated with familial amyloidotic polyneuropathy and cardiomyopathy. In most of these cases, inheritance is autosomal dominant (Reixach et al., Proc Natl Acad Sci USA, 2004, 101, 2817-2822). Jiang et al (Jiang et al., Proc Natl Acad Sci USA, 2001, 98, 14943-14948) demonstrated that the variant with a valine to isoleucine mutation at amino acid 122 (Val122Ile), which is among the most common amyloidogenic mutations worldwide, increases the velocity of rate-limiting tetramer dissociation, thereby resulting in accelerated amyloidogenesis. This finding suggests the possibility that treatments for transthyretin-related amyloidoses may include small molecules that stabilize the tetrameric form (Adamski-Werner et al., J Med Chem, 2004, 47, 355-374; Altland and Winter, Neurogenetics, 1999, 2, 183-188). Small molecule stabilizers were also shown to be of use in preventing the formation of amyloid fibrils of the wildtype transthyretin (Reixach et al., Proc Natl Acad Sci USA, 2004, 101, 2817-2822). Other common transthyretin mutations associated with amyloidosis include Val30Met and Glu61Lys. In vitro studies have shown success using ribozymes to specifically target and inhibit the expression of the Glu61Lys and Val30Met variants (Propsting et al., Biochem Biophys Res Commun, 1999, 260, 313-317; Tanaka et al., J Neurol Sci, 2001, 183, 79-84). Single-stranded oligonucleotides have been used both in vitro and in vivo to correct single-base mutation (Val30Met) in transthyretin to the wild-type form through targeted recombination (Nakamura et al., Gene Ther, 2004). The success of this therapy was limited, however, with gene conversion rates of 11% in vitro and 9% in vivo. These levels are not sufficient for suppression of the variant transthyretin in clinical terms (Nakamura et al., Gene Ther, 2004).
Thus liver transplantation is currently the only available therapy for familial amyloidotic polyneuropathy. However, this therapy is associated with several problems, and does not address conditions which are caused by transthyretin variant production in tissues other than liver (Yazaki et al., Muscle Nerve, 2003, 28, 438-442). Consequently, there remains an unmet need for agents capable of effectively modulating transthyretin expression (Nakamura et al., Gene Ther, 2004; Tanaka et al., J Neurol Sci, 2001, 183, 79-84).
The PCT publication WO200259621 and the US pre-grant publication 20020160394 claim pharmaceutical compositions for treating obesity, comprising an antisense oligonucleotide that hybridizes to a polynucleotide encoding transthyretin and reduces expression of the polynucleotide. Also claimed is the use of said oligonucleotide in the manufacture of a medicament for treating obesity (Wu, 2002).
The U.S. Pat. No. 5,744,368 discloses a primer of 22 nucleotides in length targeted to Exon 4 of transthyretin (Goldgaber et al., 1998).
Antisense technology is an effective means of reducing the expression of specific gene products and therefore is uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of transthyretin expression. The present invention provides compositions and methods for modulating transthyretin expression.